The retention of bioactive molecules in their native form is a key goal for formulation of conformationally sensitive therapeutic and diagnostic agents such as biomolecules. Moreover it is well-known that aggregated biomolecules can potentially cause adverse reactions following their administration.
To combat aggregation, it is well-known in the art that excipients can be introduced into a solution prior to drying by, for example, evaporative techniques such as freeze-drying or spray-drying in order to minimise the formation of aggregates. Excipients described in the art are proposed to play a number of roles including replacement of the water of hydration, inhibition of crystal formation in ice and other excipients, and as bulking agents. The most commonly used excipients during these drying processes are sugars such as trehalose and sucrose.
Another type of drying process involves precipitation of a bioactive molecule in a water miscible solvent. For, example preparation of bioactive molecule coated microcrystals may be carried out by coprecipitation into a water miscible organic solvent. This results in coating of a dehydrated bioactive molecule onto the surface of water soluble microcrystals. This is described in WO 00/69887, WO 2004/062560 and WO 2006/010921, which are incorporated herein by reference. Both exposure of the bioactive molecule to polar solvent and its immobilisation on a surface would be expected to expose it to significant stresses. Despite this in many cases the resultant dehydrated bioactive molecules are found to be present on the crystal surface in a native or near-native conformation. Also, it is commonly found that when the dry bioactive molecule coated microcrystals are admixed with a suitable aqueous buffer they dissolve to produce a visibly clear solution in which the reconstituted bioactive molecules are substantially in a bioactive state and have not, for example, formed significantly increased amounts of dimers, trimers and/or other soluble or insoluble aggregates.
However, it has now been discovered that certain bioactive biomolecules are much more adversely affected by precipitation and/or surface immobilisation processes. Precipitation can be promoted by a number of methods that reduce solubility such as changes in temperature and/or addition of polymers. Formation of bioactive molecule coated microcrystals requires a rapid simultaneous reduction in solubility of both the bioactive molecule and the crystalline core material. This is commonly achieved by rapid admixing with a large excess of a miscible non-solvent. An alternate method may involve admixing with a solution of different pH. It has been observed that when certain aggregation sensitive bioactive molecules are coprecipitated the resultant powders are found to have atypical properties. For example, on admixing the bioactive molecule coated microcrystals with aqueous buffer the resultant solution may contain light scattering particles that are visible to the eye or detectable by turbidity measurement techniques, known in the art. Alternatively, the solutions may appear optically clear but analysis by techniques such as size exclusion chromatography may show that a certain fraction of the soluble bioactive molecules have formed a different aggregation state from that existing in the solution used to prepare the coated microcrystals. For example, it may be found that the proportion of soluble bioactive molecules present as a monomer, dimer, trimer, tetramer or higher aggregate has increased or decreased relative to the proportion present before precipitation. For some applications the presence of changed levels of either insoluble or soluble aggregates following processing and reconstitution of bioactive molecule coated microcrystals may not present any problem, particularly if bioactive function is retained. However, in other important applications such as diagnostics or biopharmaceuticals significant changes in the level or type of aggregate will be unacceptable because they will alter, for example, concentration, bioavailability, bioactivity and/or immunogenicity. Bioactive molecules that may be found to present this problem include important diagnostic or therapeutic agents such as antibodies or cytokines and peptides such as hormones. There is a clear need, therefore, to find compositions and processes that enable such sensitive bioactive molecules to be formed into particles or coated onto microcrystals without significant changes to their aggregation state.
Problems of aggregation of bioactive biomolecules have been observed following application of other drying and particle-forming techniques such as freeze-drying, spray-drying and super-critical drying. It is therefore well established in the art that it can be advantageous to introduce one or more additives or excipients that help to protect a bioactive molecule during the steps required to carry out the drying process. Thus, for example, it is known in the art that certain excipients protect proteins in solution, certain excipients protect proteins, during freezing, certain excipients protect the protein during water removal steps such as sublimation or evaporation and certain excipients help stabilize the protein during storage in the dry state (Carpenter et al, Rational design of stable lyophilized protein formulations, Pharmaceutical Research, 1997, 14, 969-975). In addition it is known that certain combinations of excipients can be used to help to increase the solubility of dried proteins in aqueous solution (Duffy et al, Method to solubilize tissue plasminogen activator, U.S. Pat. No. 4,898,826; Kawahara et al, Modified tPA-containing injection composition having increased solubility, U.S. Pat. No. 5,425,943)
It should be further noted that the process for preparing bioactive molecule coated microcrystals uses no polymeric excipients whatsoever and involves simple mixing of an aqueous composition, in which the bioactive molecule and all of the low molecular weight components that form the particle are present and fully soluble, with an excess of a polar water miscible solvent. It is thus very different from processes such as complex coacervation where dispersions of protein or protein/polymer mixtures are solidified by contacting with immiscible organic solvents.
During the solvent coprecipitation process commonly used to form bioactive molecule coated microcrystals, the bioactive molecules will be exposed to a significantly different environment to that experienced in other techniques such as freeze-drying or spray-drying. In particular, the bioactive molecule solution will be mixed with high concentrations of a water miscible or partially water miscible organic solvent such as for example alcohols, ketones, esters or ethers or a mixture of these. These polar organic solvents will interact with a bioactive molecule in a very different way from air or vacuum and would for example, be expected to solvate the protein surface and displace water and other molecules present in the aqueous solution. In addition, during the precipitation process the bioactive molecule will have the potential to come into contact with a very high surface area formed by the microcrystalline carrier and the perceived wisdom is that exposure of molecules such as proteins to such surfaces should be avoided. Furthermore, following manufacture of precipitated particles such as bioactive molecule coated microcrystals at a commercial scale it may be necessary to store them in the organic solvent for periods ranging from many minutes to several hours or longer before they can be isolated and dried. This requires the identification of excipients that are able to protect bioactive molecules that are associated with precipitated particles against prolonged exposure to polar solvents. These conditions are clearly very different from those experienced by bioactive molecules during freeze-drying or spray drying. Therefore determining which excipients are likely to be appropriate for stabilising aggregation sensitive bioactive molecules during precipitation with polar solvents cannot be predicted from study of the prior art. Moreover, there is commercial interest in preparing particles of biomolecules by routes other than spray-drying, lyophilisation/milling or using supercritical fluids because these processes are capital expensive, often low yielding and expensive to run—precipitation methods using high concentrations of polar organic solvents are potentially more economic because they can be carried out rapidly, continuously and isothermally at or close to room temperature and at atmospheric pressure, using simple equipment. The bioactive molecule is simultaneously dehydrated and immobilised onto a particle during the process and water may be removed from the suspension simply by filtering and air drying.
However, common excipients used in spray-drying and lyophilisation such as sugars (e.g. trehalose) have unfortunately been found not to provide the necessary protection against aggregation of certain biomolecules when precipitation is carried out with polar organic solvents
It is an object of at least one aspect of the present invention to obviate or mitigate at least one or more of the aforementioned problems.
It is a further object of the present invention to maintain dehydrated bioactive molecules in their native or substantially near-native form thereby preventing or substantially preventing changes in aggregation state.